Method for controlling affinity of antibody for antigen, antibody whose affinity for antigen has been altered, and its production method

ABSTRACT

Disclosed is a method for controlling affinity of an antibody for an antigen, comprising substituting at least 3 amino acid residues of framework region 3 (FR3) defined by the Chothia method with charged amino acid residues, in an antibody whose electrical characteristic of complementarity determining region (CDR) based on the amino acid sequence of the CDR is neutral or negatively charged.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority from prior Japanese Patent ApplicationNos. 2016-255492 filed on Dec. 28, 2016 and 2017-155840 filed on Aug.10, 2017, entitled “Method for controlling affinity of antibody forantigen, antibody whose affinity for antigen has been altered, and itsproduction method”, the entire contents of which are hereby incorporatedby reference.

TECHNICAL FIELD

The present invention relates to a method for controlling affinity of anantibody for an antigen. The present invention also relates to anantibody whose affinity for an antigen has been altered and itsproduction method.

BACKGROUND

Conventionally, a technique for altering affinity of an antibody for anantigen by introducing a mutation into the amino acid sequence of theantibody has been known. For example, US 2012/0329995 A describes amethod of introducing a mutation into the amino acid sequence ofcomplementarity determining region (CDR) of an antibody to reduce theaffinity of the antibody for an antigen.

It has been also known to alter affinity for an antigen by introducing amutation into the amino acid sequence of the framework region in avariable region not into that of CDR. For example, Fukunaga A andTsumoto K, Improving the affinity of an antibody for its antigen vialong-range electrostatic interactions, Protein Eng. Des. Sel. Vol. 26,no. 12, p. 773-780, 2013 and WO 2013/084371 A describe that the 60th,63rd, 65th and 67th amino acid residues located in the framework region3 of the single chain antibody (scFv) binding to troponin I aresubstituted with a basic amino acid lysine or arginine residue. TroponinI is an antigen with a high content of charged amino acids. In FukunagaA and Tsumoto K, Improving the affinity of an antibody for its antigenvia long-range electrostatic interactions, Protein Eng. Des. Sel. Vol.26, no. 12, p. 773-780, 2013 and WO 2013/084371 A, firstly, a singlechain antibody recognizing an acidic epitope with a pI of 3.57 and asingle chain antibody recognizing a basic epitope with a pI of 11.45 areprepared. Fukunaga A and Tsumoto K, Improving the affinity of anantibody for its antigen via long-range electrostatic interactions,Protein Eng. Des. Sel. Vol. 26, no. 12, p. 773-780, 2013 and WO2013/084371 A describe that the affinity to troponin I could be improvedby utilizing the electrical attraction generated by the introduction ofbasic amino acid residues into these single chain antibodies.

SUMMARY OF THE INVENTION

The scope of the present invention is defined solely by the appendedclaims, and is not affected to any degree by the statements within thissummary.

In Fukunaga A and Tsumoto K, Improving the affinity of an antibody forits antigen via long-range electrostatic interactions, Protein Eng. Des.Sel. Vol. 26, no. 12, p. 773-780, 2013 and WO 2013/084371 A, from theviewpoint of increasing the binding rate constant in theantigen-antibody reaction, the mutation as described above is introducedin the framework region 3 (FR3) of the anti-troponin I antibody toimprove the affinity for troponin I. However, these literatures do notdescribe whether antibodies other than anti-troponin I antibody canalter affinity for an antigen by the same method.

Also, Fukunaga A and Tsumoto K, Improving the affinity of an antibodyfor its antigen via long-range electrostatic interactions, Protein Eng.Des. Sel. Vol. 26, no. 12, p. 773-780, 2013 and WO 2013/084371 Adescribe only that the affinity for an antigen has been improved. On theother hand, when using an antibody as a reagent, not only an antibodywith improved affinity for an antigen but also an antibody with reducedaffinity may be required. For example, an antibody with reduced affinityfor an antigen can be used as an appropriate control forantigen-antibody reactions. Therefore, establishment of a technique forcontrolling affinity of an antibody for an antigen is desired.

The present inventors have found that, by substituting the amino acidresidue of FR3 of an antibody with a charged amino acid residue, theaffinity for an antigen can be improved or reduced depending on the typeof the antibody. Then, the present inventors have found that suchdifference in affinity change is related to the electricalcharacteristic of CDR determined based on the number of charged aminoacid residues contained in the CDR, thereby completing the presentinvention.

Thus, a first aspect of the present invention provides a method forcontrolling affinity of an antibody for an antigen. In this method, inan antibody whose electrical characteristic of CDR based on the aminoacid sequence of the CDR is neutral or negatively charged, at least 3amino acid residues of FR3 defined by the Chothia method are substitutedwith charged amino acid residues.

Also, a second aspect of the present invention provides a method forproducing an antibody whose affinity for an antigen has been altered.This method comprises the steps of substituting at least 3 amino acidresidues of FR3 defined by the Chothia method with a charged amino acidresidue in an antibody whose electrical characteristic of CDR based onthe amino acid sequence of the CDR is neutral or negatively charged, andrecovering the antibody obtained in the substitution step.

Furthermore, a third aspect of the present invention provides anantibody whose affinity for an antigen has been altered. In thisantibody, the electrical characteristic of CDR based on the amino acidsequence of the CDR is neutral or negatively charged, and at least 3amino acid residues of FR3 defined by the Chothia method in theunmodified antibody are substituted with charged amino acid residues.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A is a graph showing a dissociation constant in an interactionbetween a wild-type anti-insulin antibody and its variant, and anantigen (insulin);

FIG. 1B is a graph showing a dissociation constant in an interactionbetween a wild-type anti-thyroid stimulating hormone receptor (TSHR)antibody and its variant, and an antigen (TSHR);

FIG. 2A is a diagram showing the surface charge distribution of awild-type anti-insulin antibody and its variant, and an antigen(insulin);

FIG. 2B is a diagram showing the surface charge distribution of awild-type anti-TSHR antibody and its variant, and an antigen (TSHR);

FIG. 3 is a graph showing analytical peaks when the thermal stability ofa wild-type anti-insulin antibody and its variant is measured by adifferential scanning calorimeter (DSC);

FIG. 4 is a graph showing a dissociation constant in an interactionbetween a wild-type anti-lysozyme antibody and its variant, and anantigen (lysozyme); and

FIG. 5 is a graph showing a dissociation constant in an interactionbetween a wild-type anti-hepatitis B surface antigen (HBsAg) antibodyand its variant, and an antigen (HBsAg).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 1. Method forControlling Affinity of Antibody for Antigen

In the method for controlling affinity of an antibody for an antigen ofthe present embodiment (hereinafter, also referred to as “controlmethod”), an antibody whose electrical characteristic of CDR based onthe amino acid sequence of the CDR is neutral or negatively charged isfor controlling the affinity for an antigen. In the control method ofthe present embodiment, in the antibody having such electricalcharacteristic, it is possible to control affinity of the antibody foran antigen by substituting at least 3 amino acid residues of FR3 definedby the Chothia method with charged amino acid residues. As used herein,the phrase “controlling affinity” refers to both improving the affinityof an antibody for an antigen and reducing the affinity of an antibodyfor an antigen. Therefore, the control method of the present embodimentmay be interpreted as a method of altering affinity of an antibody foran antigen.

In the control method of the present embodiment, the original antibodyfor controlling the affinity for an antigen is also referred to as“unmodified antibody”. Herein, substituting the amino acid residue ofFR3 defined by the Chothia method in an unmodified antibody with acharged amino acid residue is also referred to as “introducing amutation”. Such substitution is also referred to as “introduction ofmutation” or simply “mutation”. An antibody obtained by introducing amutation into an unmodified antibody is also referred to as “an antibodywhose affinity is controlled”.

In the present embodiment, the surface charge distribution of theunmodified antibody is changed by the introduction of mutation, and theaffinity for an antigen is controlled. That is, the antibody whoseaffinity is controlled has improved or reduced affinity for an antigenas compared to the unmodified antibody. In the present embodiment, theaffinity of the antibody whose affinity is controlled for an antigen maybe evaluated by a kinetic parameter in an antigen-antibody reaction ormay be evaluated by an immunological measurement method such as an ELISAmethod. The kinetic parameter includes binding rate constant (k_(on)),dissociation rate constant (k_(off)) and dissociation constant (K_(D)),and is preferably K_(D). The kinetic parameter in an antigen-antibodyreaction can be obtained by surface plasmon resonance (SPR) technology.

In the case where the affinity of the antibody for an antigen isimproved by the control method of the present embodiment, for example,the value of K_(D) in the antigen-antibody reaction is about ½, about ⅕,about 1/10, about 1/20, about 1/50, about 1/100 or about 1/1000, ascompared to the unmodified antibody. On the other hand, when theaffinity of the antibody for an antigen is reduced, the value of K_(D)in the antigen-antibody reaction is about 2 times, about 5 times, about10 times, about 20 times, about 50 times, about 100 times or about 1000times, as compared to the unmodified antibody.

In the present embodiment, the unmodified antibody may be an antibodyrecognizing any antigen. In a preferred embodiment, the unmodifiedantibody is an antibody in which the base sequence of genes encoding thevariable regions of light chain and heavy chain is known or an antibodyin which the base sequence can be confirmed. Specifically, it is anantibody in which the base sequence of the antibody gene is disclosed ina known database, or an antibody in which the hybridoma producing theantibody is available. Examples of such database include GeneBankprovided by National Center for Biotechnology Information (NCBI) and thelike. The antibody class may be IgG, IgA, IgM, IgD or IgE, and ispreferably IgG.

In the present embodiment, the unmodified antibody may be in the form ofan antibody fragment as long as it has a variable region including FR3to which a mutation is to be introduced. The antibody whose affinity iscontrolled may be in the form of an antibody fragment as long as it hasa variable region including FR3 into which a mutation has beenintroduced. Examples of such antibody fragment include Fab fragments,F(ab′)2 fragments, Fab′ fragments, Fd fragments, Fv fragments, dAbfragments, single chain antibodies (scFv), and the like. Among them, Fabfragments are particularly preferred.

Three CDRs are present in each variable region of the light chain andheavy chain of the antibody and constitute the antigen-binding site ofthe antibody. The three CDRs are called CDR1, CDR2 and CDR3, countingfrom the amino terminus of the antibody chain. Since the CDR is involvedin the specificity of the antibody, in the present embodiment, it ispreferable that the antibody whose affinity is controlled does not havea mutation in the CDR. That is, the amino acid sequence of the CDR ofthe antibody whose affinity is controlled is preferably the same as theamino acid sequence of the CDR of the unmodified antibody.

The framework region (FR) is a region other than the CDRs present ineach variable region of the light chain and heavy chain of the antibody.FR plays a role of a scaffold linking the three CDRs and contributes tothe structural stability of the CDR. Therefore, the amino acid sequenceof FR is highly conserved between antibodies of the same species. FR3 isone of FRs, and refers to the region between CDR2 and CDR3.

In the art, a method of numbering the amino acid residues of the CDR(hereinafter, also referred to as “numbering method”) for defining theboundary and length of the CDR is known. As such numbering method, forexample, the Chothia method (Chothia C. and Lesk A M., CanonicalStructures for the Hypervariable Regions of Immunoglobulins., J MolBiol., vol. 196, p. 901-917, 1987), the Kabat method (Kabat E A. et al.,Sequences of Proteins of Immunological Interest., NIH publication No.91-3242), the IMGT method (Lefranc MP., IMGT Unique Numbering for theVariable (V), Constant (C), and Groove (G) Domains of IG, TR, MH, IgSF,and MhSF., Cold Spring Harb Protoc. 2011(6): 633-642, 2011), theHonegger method (Honegger A. et al., Yet Another Numbering Scheme forImmunoglobulin Variable Domains: An Automatic Modeling and AnalysisTool., J Mol Biol., vol. 309, p. 657-670, 2001), the ABM method, theContact method and the like are known. When numbers are assigned to theamino acid residues of the CDRs, the FR that is a region other than theCDRs is also numbered. In the present embodiment, the boundary andlength of CDR and FR3 are defined by the Chothia method, but they canalso be defined by other numbering methods.

In the Chothia method, light chain FR3 is defined as a region consistingof amino acid residues 53 to 90, and heavy chain FR3 is defined as aregion consisting of amino acid residues 56 to 95. Here, for comparison,the numbers of the light chain FR3 and heavy chain FR3 (the positions ofthe amino acid residues at the start and end points of FR3) as definedby the Chothia method and other numbering methods are shown in Tables 1and 2. The Vernier zone residue in the table is an amino acid residuecontributing to the structural stability of the CDR among the amino acidresidues contained in the FR. Tables 1 and 2 also show the positions ofthe Vernier zone residues in FR3, as defined by the numbering methods.Table 1 also shows the positions where mutation was introduced in FR3 ofthe light chain in Example 1, as defined by the numbering methods.

TABLE 1 Position where mutation Numbering Light was introduced in lightmethod chain FR3 Vernier zone residue chain FR3 in Example 1 Chothia53-90 64, 66, 68, 69, 71 63, 65, 67, 70, 72 Kabat 57-88 64, 66, 68, 69,71 63, 65, 67, 70, 72 IMGT  66-104 78, 80, 84, 85, 87 77, 79, 83, 86, 88Honergger  78-108 80, 82, 84, 87, 89 79, 81, 83, 88, 90 ABM 57-88 64,66, 68, 69, 71 63, 65, 67, 70, 72 Contact 56-88 64, 66, 68, 69, 71 63,65, 67, 70, 72

TABLE 2 Numbering Heavy method chain FR3 Vernier zone residue Chothia56-98 67, 69, 71, 73, 78, 93, 94 Kabat 66-94 67, 69, 71, 73, 78, 93, 94IMGT  66-104 76, 78, 80, 82, 87 Honergger  78-108 78, 80, 82, 84, 89,107, 108 ABM 59-94 67, 69, 71, 73, 78, 93, 94 Contact 59-92 67, 69, 71,73, 78

In the present embodiment, at least three mutations may be introducedinto any of the amino acid residues of FR3 defined by the Chothia method(hereinafter, also simply referred to as “FR3”) in an unmodifiedantibody. Preferably, at least three mutations are introduced into aminoacid residues in the region excluding amino acid residues that arefolded into the interior of the molecule from FR3 and are not exposed tothe surface (hereinafter, also referred to as “unexposed residues”). Itis expected that, even when a mutation is introduced into an unexposedresidue, it will not affect the surface charge, so it is preferable toexclude an unexposed residue from the position where a mutation is to beintroduced. Specifically, the amino acid residues in the regionexcluding the unexposed residues from FR3 are the 53rd to 81st aminoacid residues of the FR3 of the light chain, and the 56th to 88th aminoacid residues of the FR3 of the heavy chain.

More preferably, at least three mutations are introduced into the aminoacid residues in the region excluding the unexposed residues and theVernier zone residues from FR3. As described above, it is because theVernier zone residue contributes to the structural stability of the CDR.Specifically, the amino acid residues in the region excluding theunexposed residues and the Vernier zone residue from FR3 are the 53rd to63rd, 65th, 67th, 70th and 72nd to 81st amino acid residues of the FR3of the light chain, and the 56th to 66th, 68th, 70th, 72nd, 74th to 77thand 79th to 88th amino acid residues of the FR3 of the heavy chain.

Particularly more preferably, at least three mutations are introducedinto amino acid residues whose side chains are oriented toward themolecular surface, among the amino acid residues in the region excludingthe unexposed residues and the Vernier zone residues from FR3. The aminoacid residues whose side chains are oriented toward the molecularsurface are substituted with charged amino acid residues, whereby thecontribution to the surface charge becomes larger. The amino acidresidues whose side chains are oriented toward the molecular surface inFR3 refer to the 53rd, 54th, 56th, 57th, 60th, 63rd, 65th, 67th, 70th,72nd, 74th, 76th, 77th and 79th to 81st amino acid residues of the FR3of the light chain, and the 56th, 57th, 59th, 61st, 62nd, 64th to 66th,68th, 70th, 72nd, 74th, 75th, 77th, 79th, 81st, 83rd, 84th and 86th to88th amino acid residues of the FR3 of the heavy chain.

In the present embodiment, at least three mutations may be introduced ineither the FR3 of the light chain or the FR3 of the heavy chain. Fromthe viewpoint of thermal stability of the antibody, it is preferable tointroduce at least three mutations in the FR3 of the light chain. Whenthe FR3s of both the light chain and the heavy chain have a mutation, itis preferable to introduce at least three mutations in the FR3 of thelight chain and introduce at least three mutations in the FR3 of theheavy chain.

In the present embodiment, the upper limit of the number of mutationsintroduced in FR3 is not particularly limited, but is preferably 16amino acids or less. That is, the number of mutations in FR3 of theantibody whose affinity is controlled is specifically 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, or 16.

As described above, in the control method of the present embodiment, thesurface charge distribution changes due to the introduction of mutationinto the unmodified antibody, and the affinity for an antigen changes.Thus, it is preferred that at least all three mutations aresubstitutions with charged amino acid residues having the same charge.That is, it is preferred that at least all three mutations aresubstitutions at acidic amino acid residues or substitutions at basicamino acid residues.

The charged amino acid residue refers to an aspartic acid residue, aglutamic acid residue, a lysine residue, an arginine residue, and ahistidine residue. The acidic amino acid residue refers to an asparticacid residue and a glutamic acid residue. The basic amino acid residuerefers to a lysine residue, an arginine residue, and a histidineresidue. In the present embodiment, as a basic amino acid residue to beintroduced in FR3 as a mutation, a lysine residue and an arginineresidue are preferable.

In the present embodiment, at least three mutations to be introducedinto the unmodified antibody may be a mutation that substitutes theneutral amino acid residue of FR3 with a charged amino acid residue. Theneutral amino acid residues refer to an alanine residue, an asparagineresidue, an isoleucine residue, a glycine residue, a glutamine residue,a cysteine residue, a threonine residue, a serine residue, a tyrosineresidue, a phenylalanine residue, a proline residue, a valine residue, amethionine residue, a leucine residue, and a tryptophan residue.

As described above, in the present embodiment, an antibody whoseelectrical characteristic of CDR based on the amino acid sequence of theCDR is neutral or negatively charged is for controlling the affinity foran antigen. Herein, the electrical characteristic of CDR is an indexuniquely defined by the present inventors. The electrical characteristicof CDR is determined based on the number of charged amino acid residuesin the amino acid sequence of the CDR. Specifically, the electricalcharacteristic of CDR is determined by the following formula (I).

X=[Number of basic amino acid residues in amino acid sequence ofCDR]−[Number of acidic amino acid residues in amino acid sequence ofCDR]  (I)

wherein when X is −1, 0 or 1, the electrical characteristic of CDR isneutral,

when X is 2 or more, the electrical characteristic of CDR is positivelycharged, and

when X is −2 or less, the electrical characteristic of CDR is negativelycharged.

The electrical characteristic of CDR is preferably determined based onthe amino acid sequences of the CDRs of both the light chain and theheavy chain. In this case, the amino acid sequence of the CDR in theformula (I) refers to all amino acid sequences of CDR1, CDR2 and CDR3 ofthe light chain and CDR1, CDR2 and CDR3 of the heavy chain. In thepresent embodiment, it is preferable to substitute at least 3 amino acidresidues of the FR3 of the light chain with charged amino acid residues,in an antibody whose electrical characteristic determined based on theamino acid sequences of the CDRs of both the light chain and the heavychain is neutral or negatively charged.

The electrical characteristic of CDR may be determined for each of thelight chain CDR and the heavy chain CDR. That is, when determining theelectrical characteristic of the light chain CDR, the amino acidsequence of the CDR in the formula (I) refers to all amino acidsequences of CDR1, CDR2 and CDR3 of the light chain. When determiningthe electrical characteristic of the heavy chain CDR, the amino acidsequence of the CDR in the formula (I) refers to all amino acidsequences of CDR1, CDR2 and CDR3 of the heavy chain. In the presentembodiment, it is preferable to substitute at least 3 amino acidresidues of the FR3 of the light chain with charged amino acid residues,in an antibody whose electrical characteristic of the light chain CDR isneutral or negatively charged.

The amino acid sequence of the CDR can be obtained from a publicdatabase that discloses the sequence of the antibody gene.Alternatively, when there is a hybridoma that produces an unmodifiedantibody, the amino acid sequence of the CDR can be obtained byobtaining a nucleic acid encoding a heavy chain and a light chain fromthe hybridoma by a known method, and sequencing the base sequence of thenucleic acid.

The electrical characteristic of CDR differs depending on the antibody.For example, as shown in Example 3 described below, the light chain CDRof a wild-type (i.e., unmodified) anti-insulin antibody has one basicamino acid residue (arginine), and no acidic amino acid residue exists.Thus, the electrical characteristic of CDR of the wild-type anti-insulinantibody is defined as neutral (X=1). The light chain CDR of a wild-typeanti-TSHR antibody has five acidic amino acid residues (aspartic acid),and no basic amino acid residue exists. Thus, the electricalcharacteristic of CDR of the wild-type anti-TSHR antibody is defined asnegatively charged (X=−5).

In an unmodified antibody whose electrical characteristic of CDR isneutral, by substituting at least 3 amino acid residues of FR3 withacidic amino acid residues, a wide range of surface charges includingthe antigen-binding site of the antibody becomes negative. In addition,in an unmodified antibody whose electrical characteristic of CDR isneutral, by substituting at least 3 amino acid residues of FR3 withbasic amino acid residues, a wide range of surface charges including theantigen-binding site of the antibody becomes positive. By such a changein the surface charge, electrostatic interaction (attraction orrepulsion) is generated when the antibody and the antigen bind. That is,in an unmodified antibody whose electrical characteristic of CDR isneutral, by substituting at least 3 amino acid residues of FR3 withacidic amino acid residues, the affinity of the antibody for an antigencan be reduced as compared to that of the unmodified antibody. Inaddition, in an unmodified antibody whose electrical characteristic ofCDR is neutral, by substituting at least 3 amino acid residues of FR3with basic amino acid residues, the affinity of the antibody for anantigen can be improved as compared to that of the unmodified antibody.

In an antibody whose electrical characteristic of CDR is negativelycharged, by substituting at least 3 amino acid residues of FR3 withacidic amino acid residues, a wide range of surface charges includingthe antigen-binding site of the antibody becomes negative. By such achange in the surface charge, electrostatic interaction (repulsion) isgenerated when the antibody and the antigen bind. That is, in anunmodified antibody whose electrical characteristic of CDR is negativelycharged, by substituting at least 3 amino acid residues of FR3 withacidic amino acid residues, the affinity of the antibody for an antigencan be reduced as compared to that of the unmodified antibody.

In the present embodiment, a mutation can be introduced in FR3 of theunmodified antibody by known methods such as DNA recombinationtechnology and other molecular biological techniques. For example, whenthere is a hybridoma that produces an unmodified antibody, as shown inExample 1 described later, RNA extracted from the hybridoma is used tosynthesize each of a polynucleotide encoding the light chain and apolynucleotide encoding the heavy chain, by a reverse transcriptionreaction and a RACE (Rapid Amplification of cDNA ends) method. Thesepolynucleotides are amplified by PCR using primers for introducing amutation into at least 3 amino acid residues of FR3 to obtain apolynucleotide encoding the light chain into which a mutation has beenintroduced in FR3 and a polynucleotide encoding the heavy chain intowhich a mutation has been introduced in FR3. The obtained polynucleotideis incorporated into an expression vector known in the art to obtain anexpression vector containing a polynucleotide encoding an antibody whoseaffinity is controlled. Here, the polynucleotide encoding the lightchain and the polynucleotide encoding the heavy chain may beincorporated into one expression vector or may be separatelyincorporated into two expression vectors. The type of the expressionvector is not particularly limited, and it may be an expression vectorfor mammalian cells or an expression vector for E. coli. By transducingor transfecting the obtained expression vector into an appropriate hostcell (for example, mammalian cell or E. coli), an antibody whoseaffinity is controlled can be obtained.

When obtaining an antibody whose affinity is controlled which is asingle chain antibody (scFv), as shown in, for example, WO 2013/084371A, RNA extracted from the hybridoma may be used to synthesize each of apolynucleotide encoding a light chain variable region and apolynucleotide encoding a heavy chain variable region by a reversetranscription reaction and PCR. These polynucleotides are ligated byoverlap extension PCR or the like to obtain a polynucleotide encoding anunmodified scFv. The obtained polynucleotide is amplified by PCR using aprimer for introducing a mutation into at least 3 amino acid residues ofFR3 to obtain a polynucleotide encoding scFv into which a mutation hasbeen introduced in FR3. The obtained polynucleotide is incorporated intoan expression vector known in the art to obtain an expression vectorcontaining a polynucleotide encoding an antibody whose affinity iscontrolled in the form of scFv. By transducing or transfecting theobtained expression vector into an appropriate host cell, an antibodywhose affinity is controlled in the form of scFv can be obtained.

When there is no hybridoma that produces an antibody that recognizes anantigen of interest, an antibody-producing hybridoma may be prepared byknown methods such as those described in, for example, Kohler andMilstein, Nature, vol. 256, p. 495-497, 1975. Alternatively, RNAobtained from the spleen of an animal such as a mouse immunized with anantigen of interest may be used. When the RNA obtained from the spleenis used, for example, as shown in Fukunaga A and Tsumoto K, Improvingthe affinity of an antibody for its antigen via long-range electrostaticinteractions, Protein Eng. Des. Sel. Vol. 26, no. 12, p. 773-780, 2013,a polynucleotide encoding an unmodified Fab having desired affinity maybe selected from among the polynucleotides encoding the obtainedunmodified Fab by phage display method or the like.

2. Method for Producing Antibody whose Affinity for Antigen has beenAltered

The scope of the present disclosure also includes a method for producingan antibody (hereinafter, also referred to as “production method”) whoseaffinity for an antigen has been altered. In the production method ofthe present embodiment, first, in an antibody whose electricalcharacteristic of CDR based on the amino acid sequence of the CDR isneutral or negatively charged, a step of substituting at least 3 aminoacid residues of FR3 defined by the Chothia method with charged aminoacid residues is carried out.

In the production method of the present embodiment, the antibody inwhich the amino acid residue of FR3 is substituted is the same as theunmodified antibody in the control method of the present embodiment.Hereinafter, in the production method of the present embodiment, theoriginal antibody for altering affinity for an antigen is also referredto as “unmodified antibody”. The details of the electricalcharacteristic of CDR are the same as those described for the controlmethod of the present embodiment. The electrical characteristic of CDRof the antibody can be determined by the above formula (I). FR3 definedby the Chothia method is the same as that described for the controlmethod of the present embodiment and is as shown in Tables 1 and 2.

In the production method of the present embodiment, it is possible toobtain an antibody whose affinity for an antigen has been altered bychanging the surface charge distribution of the antibody by substitutionof an amino acid residue. That is, it can be said that the abovesubstitution step is, in the antibody whose electrical characteristic ofCDR is neutral or negatively charged, a step of substituting at least 3amino acid residues of FR3 defined by the Chothia method with chargedamino acid residues to alter affinity of the antibody for an antigen.For example, the above substitution step may be, in an antibody whoseelectrical characteristic of CDR is neutral, a step of substituting atleast 3 amino acid residues of FR3 defined by the Chothia method withacidic amino acid residues to reduce affinity of the antibody for anantigen as compared to that of the unmodified antibody. The abovesubstitution step may be, in an antibody whose electrical characteristicof CDR is neutral, a step of substituting at least 3 amino acid residuesof FR3 defined by the Chothia method with basic amino acid residues toimprove affinity of the antibody for an antigen as compared to that ofthe unmodified antibody. Alternatively, the above substitution step maybe, in an antibody whose electrical characteristic of CDR is negativelycharged, a step of substituting at least 3 amino acid residues of FR3defined by the Chothia method with acidic amino acid residues to reduceaffinity of the antibody for an antigen as compared to that of theunmodified antibody.

In the present embodiment, it is preferable to substitute at least 3amino acid residues of the FR3 of the light chain with charged aminoacid residues, in an antibody whose electrical characteristic determinedbased on the amino acid sequences of the CDRs of both the light chainand the heavy chain is neutral or negatively charged. It is preferableto substitute at least 3 amino acid residues of the FR3 of the lightchain with charged amino acid residues, in an antibody whose electricalcharacteristic of the light chain CDR is neutral or negatively charged.

Substitution of an amino acid residue can be carried out by knownmethods such as DNA recombination technology and other molecularbiological techniques. For example, when there is a hybridoma thatproduces an antibody whose electrical characteristic of CDR is neutralor negatively charged, an expression vector containing a polynucleotideencoding an antibody whose affinity for an antigen has been altered isdetermined can be obtained in the same manner as described for thecontrol method of the present embodiment. Moreover, by transducing ortransfecting the obtained expression vector into an appropriate hostcell, a host cell expressing the antibody can be obtained.

Then, in the production method of the present embodiment, the antibodyobtained in the above substitution step is recovered. For example, ahost cell expressing an antibody whose affinity for an antigen has beenaltered is dissolved in a solution containing an appropriate solubilizerto liberate the antibody in the solution. When the above host cellsecretes an antibody whose affinity for an antigen has been altered intothe medium, the culture supernatant is recovered. The liberated antibodycan be recovered by methods known in the art such as affinitychromatography. For example, when the produced antibody is IgG, theantibody can be recovered by affinity chromatography using protein A orG. If necessary, the recovered antibody may be purified by methods knownin the art such as gel filtration.

The affinity of the prepared antibody for an antigen may be evaluated bya kinetic parameter in an antigen-antibody reaction or may be evaluatedby an immunological measurement method such as an ELISA method. In anantibody where the affinity for an antigen is improved, for example, thevalue of K_(D) in the antigen-antibody reaction is about ½, about ⅕,about 1/10, about 1/20, about 1/50, about 1/100 or about 1/1000, ascompared to the original antibody. In an antibody where the affinity foran antigen is reduced, the value of K_(D) in the antigen-antibodyreaction is about 2 times, about 5 times, about 10 times, about 20times, about 50 times, about 100 times or about 1000 times, as comparedto the original antibody.

3. Antibody whose Affinity for Antigen has been Altered

The scope of the present disclosure also includes an antibody whoseaffinity for an antigen has been altered (hereinafter, also referred toas “modified antibody”). The modified antibody of the present embodimentis characterized in that the electrical characteristic of CDR based onthe amino acid sequence of the CDR is neutral or negatively charged. Thedetails of the electrical characteristic of CDR are the same as thosedescribed for the control method of the present embodiment. Theelectrical characteristic of CDR of the modified antibody can bedetermined by the above formula (I).

In the modified antibody of the present embodiment, at least 3 aminoacid residues of FR3 defined by the Chothia method in an unmodifiedantibody are substituted with charged amino acid residues. That is, themodified antibody of the present embodiment has at least three mutationsdue to substitution with charged amino acid residues in FR3 defined bythe Chothia method, as compared to the amino acid sequence of theunmodified antibody. The modified antibody of the present embodiment isthe same as the above-described “antibody whose affinity is controlled”.FR3 defined by the Chothia method is the same as that described for thecontrol method of the present embodiment and is as shown in Tables 1 and2.

Here, the unmodified antibody refers to an antibody before the affinityfor an antigen is altered. That is, the unmodified antibody is theoriginal antibody of the modified antibody, and the amino acid residueof FR3 defined by the Chothia method is not substituted with a chargedamino acid residue. This unmodified antibody corresponds to the originalantibody for controlling the affinity for an antigen in the controlmethod of the present embodiment. In the present embodiment, theunmodified antibody has a CDR whose electrical characteristic is neutralor negatively charged.

In the modified antibody of the present embodiment, the surface chargedistribution of the antibody is changed by the introduction of mutation.That is, the affinity of the modified antibody for an antigen isimproved or reduced as compared to that of the unmodified antibody. Inthe present embodiment, the affinity of the modified antibody for anantigen may be evaluated by a kinetic parameter in an antigen-antibodyreaction or may be evaluated by an immunological measurement method suchas an ELISA method. The type and acquisition of the kinetic parameterare the same as those described for the control method of the presentembodiment.

In a modified antibody where the affinity for an antigen is improved,for example, the value of K_(D) in the antigen-antibody reaction isabout ½, about ⅕, about 1/10, about 1/20, about 1/50, about 1/100 orabout 1/1000, as compared to the unmodified antibody. In a modifiedantibody where the affinity for an antigen is reduced, for example, thevalue of K_(D) in the antigen-antibody reaction is about 2 times, about5 times, about 10 times, about 20 times, about 50 times, about 100 timesor about 1000 times, as compared to the unmodified antibody.

The modified antibody may be an antibody recognizing any antigen. Theantibody class may be IgG, IgA, IgM, IgD or IgE, and is preferably IgG.The modified antibody of the present embodiment may be in the form of anantibody fragment as long as it has a variable region including FR3 intowhich a mutation has been introduced. The type of the antibody fragmentis the same as that described for the control method of the presentembodiment.

It is preferable that the modified antibody of the present embodimentdoes not have a mutation in the CDR. That is, the amino acid sequence ofthe CDR of the modified antibody is preferably the same as the aminoacid sequence of the CDR of the unmodified antibody.

In the modified antibody of the present embodiment, at least threemutations may be introduced into any amino acid residue of FR3 definedby the Chothia method in the unmodified antibody. Preferably, at leastthree mutations are introduced into the amino acid residues in theregion excluding the unexposed residues from FR3. The amino acidresidues in the region excluding the unexposed residues from FR3 are thesame as those described for the control method of the presentembodiment.

More preferably, at least three mutations are introduced into the aminoacid residues in the region excluding the unexposed residues and theVernier zone residues from FR3. The amino acid residues in the regionexcluding the unexposed residues and the Vernier zone residues from FR3are the same as those described for the control method of the presentembodiment.

Particularly more preferably, at least three mutations are introducedinto amino acid residues whose side chains are oriented toward themolecular surface, among the amino acid residues in the region excludingthe unexposed residues and the Vernier zone residues from FR3. The aminoacid residues whose side chains are oriented toward the molecularsurface in FR3 are the same as those described for the control method ofthe present embodiment.

In the present embodiment, mutations of at least 3 amino acid residuesmay be introduced into either the FR3 of the light chain or the FR3 ofthe heavy chain, but is preferably introduced into the FR3 of the lightchain. When the FR3s of both the light chain and the heavy chain have amutation, it is preferable to introduce mutations of at least 3 aminoacid residues into the FR3 of the light chain and mutations of at least3 amino acid residues into the FR3 of the heavy chain.

In the present embodiment, the upper limit of the number of mutations inFR3 of the modified antibody is not particularly limited, but ispreferably 16 amino acids or less. Specifically, the number of mutationsin FR3 of the modified antibody is 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15 or 16.

In the present embodiment, at least three mutations of the modifiedantibody may be mutations in which the neutral amino acid residues ofFR3 are substituted with charged amino acid residues. In the presentembodiment, it is preferred that at least all three mutations aresubstitutions with charged amino acid residues having the same charge.That is, it is preferred that at least all three mutations aresubstitutions at acidic amino acid residues or substitutions at basicamino acid residues.

Examples of the modified antibody of the present embodiment include thefollowing antibodies (1) to (3).

(1) An antibody whose electrical characteristic of CDR is neutral, atleast 3 amino acid residues of FR3 defined by the Chothia method havebeen substituted with acidic amino acid residues, and the affinity ofthe antibody for an antigen is reduced as compared to that of anunmodified antibody,

(2) An antibody whose electrical characteristic of CDR is neutral, atleast 3 amino acid residues of FR3 defined by the Chothia method havebeen substituted with basic amino acid residues, and the affinity of theantibody for an antigen is improved as compared to that of an unmodifiedantibody, and

(3) An antibody whose electrical characteristic of CDR is negativelycharged, at least 3 amino acid residues of FR3 defined by the Chothiamethod have been substituted with acidic amino acid residues, and theaffinity of the antibody for an antigen is reduced as compared to thatof an unmodified antibody.

Specific examples of the modified antibody include modified antibodiesof anti-insulin antibodies and anti-TSHR antibodies. In such a modifiedantibody of anti-insulin antibody, the electrical characteristic of CDRis neutral, and the 63rd, 65th, 67th, 70th and 72nd amino acid residuesin the FR3 of the light chain are substituted with basic amino acidresidues. In this modified antibody, the affinity for insulin, which isan antigen, is improved as compared to the unmodified antibody. On theother hand, in a modified antibody in which the 63rd, 65th, 67th, 70thand 72nd amino acid residues in the FR3 of the light chain have beensubstituted with acidic amino acid residues, the affinity for insulin,which is an antigen, is reduced as compared to the unmodified antibody.In the modified antibody of anti-TSHR antibody, the electricalcharacteristic of CDR is negatively charged, and the 63rd, 65th, 67th,70th and 72nd amino acid residues in the FR3 of the light chain aresubstituted with acidic amino acid residues. In this modified antibody,the affinity for TSHR, which is an antigen, is reduced as compared tothe unmodified antibody.

The modified antibody of the present embodiment can be prepared by knownmethods such as DNA recombination technology and other molecularbiological techniques. For example, when there is a hybridoma thatproduces an antibody whose electrical characteristic of CDR is neutralor negatively charged, an antibody in which at least 3 amino acidresidues of FR3 are substituted with charged amino acid residues can beprepared in the same manner as those described for the control methodand the production method of the present embodiment.

In the present embodiment, the use method of the modified antibody isnot particularly different from that of the unmodified antibody. Themodified antibody can be used for various tests, research and the like,as well as the unmodified antibody. The modified antibody of the presentembodiment may be modified with a labeling substance or the like knownin the art.

The scope of the present disclosure also includes isolated and purifiedpolynucleotides encoding an antibody whose affinity for an antigen hasbeen altered of the present embodiment or fragments thereof. It ispreferred that the isolated and purified polynucleotide encoding thefragment of the modified antibody of the present embodiment encodes avariable region including FR3 into which a mutation has been introduced.The scope of the present disclosure also includes a vector containingthe above polynucleotide. A vector is a polynucleotide constructdesigned for transduction or transfection. The type of vector is notparticularly limited. The vector can be appropriately selected fromvectors known in the art such as expression vectors, cloning vectors,viral vectors and the like. The scope of the present disclosure alsoincludes a host cell containing the vector. The type of the host cell isnot particularly limited. The host cell can be appropriately selectedfrom eukaryotic cells, prokaryotic cells, mammalian cells and the like.

Hereinafter, the present disclosure will be described in more detail byexamples, but the present disclosure is not limited to these examples.

EXAMPLES Example 1 Preparation of Antibody into which Charged Amino AcidResidue Introduced in FR3

Variants of each antibody were prepared by substituting 3 or 5 aminoacid residues of FR3 of anti-insulin antibody andanti-thyroid-stimulating hormone receptor (TSHR) antibody with chargedamino acid residues.

(1) Acquisition of Genes of Wild-Type Anti-Insulin Antibody andWild-Type Anti-TSHR Antibody Reagents

ISOGEN (NIPPON GENE CO., LTD.)

SMARTer (registered trademark) RACE 5′/3′ kit (clontech)

10×A-attachment mix (TOYOBO CO., LTD.)

pcDNA (trademark) 3.4 TOPO (registered trademark) TA cloning kit (ThermoFisher Scientific K.K.)

Competent high DH5α (TOYOBO CO., LTD.)

QIAprep Spin Miniprep kit (QIAGEN)

KOD plus neo (TOYOBO CO., LTD.)

Ligation high ver. 2 (TOYOBO CO., LTD.)

(1.1) Acquisition of Wild-Type Anti-Insulin Antibody Gene (1.1.1)Extraction of Total RNA from Antibody-Producing Hybridoma

Hybridomas that produce a wild-type mouse anti-human insulin antibodywere prepared by using human insulin as an antigen, according to themethod described in Kohler and Milstein, Nature, vol. 256, p. 495-497,1975. The hybridoma culture (10 mL) was centrifuged at 1000 rpm for 5minutes, then the supernatant was removed. The resulting cells weredissolved with ISOGEN (1 mL). The solution was allowed to stand at roomtemperature for 5 minutes. Chloroform (200 μL) was added thereto, andthe mixture was stirred for 15 seconds. Then, the mixture was allowed tostand at room temperature for 3 minutes. Then, this was centrifuged at12000×G at 4° C. for 10 minutes, and an aqueous phase (500 μL)containing RNA was recovered. Isopropanol (500 μL) was added to therecovered aqueous phase, and the mixture was mixed. The resultingmixture was allowed to stand at room temperature for 5 minutes.Thereafter, the resulting mixture was centrifuged at 12000×G at 4° C.for 10 minutes. The supernatant was removed, and 70% ethanol (1 mL) wasadded to the resulting precipitate (total RNA). The mixture wascentrifuged at 7500×G at 4° C. for 10 minutes. The supernatant wasremoved, and RNA was air-dried. The RNA was dissolved in RNase-freewater (20 μL).

(1.1.2) Synthesis of cDNA

Using each of the total RNAs obtained in the above (1.1.1), RNA sampleshaving the following composition were prepared.

RNA Sample

Total RNA (500 ng/μL) 1 μL RT Primer 1 μL Deionized water 1.75 μL Total3.75 μL

The prepared RNA sample was heated at 72° C. for 3 minutes. Thereafter,the RNA sample was incubated at 42° C. for 2 minutes. Then, a cDNAsynthesis sample was prepared by adding 12 μM SMARTer II Aoligonucleotide (1 μL) to the RNA sample. Using this cDNA synthesissample, a reverse transcription reaction solution having the followingcomposition was prepared.

Reverse Transcription Reaction Solution

5x First-Strand buffer 2 μL 20 mM DTT 1 μL 10 mM dNTP mix 1 μL RNaseinhibitor 0.25 μL SMARTScribe RT(100 U/μL) 1 μL cDNA synthesis sample4.75 μL Total 10 μL

The prepared reverse transcription reaction solution was reacted at 42°C. for 90 minutes. Then, the reaction solution was heated at 70° C. for10 minutes, and tricine-EDTA (50 μL) was added thereto. Using theobtained solution as a cDNA sample, a 5′RACE reaction solution havingthe following composition was prepared.

[5′RACE Reaction Solution]

10x PCR buffer 5 μL dNTP mix 5 μL 25 mM Mg₂SO₄ 3.5 μL cDNA sample 2.5 μL10x Universal Primer Mix 5 μL 3′-Primer 1 μL KOD plus neo (1 U/μL) 1 μLPurified water 27 μL Total 50 μL

The prepared 5′RACE reaction solution was subjected to RACE reactionunder the following reaction conditions. The following “Y” is 90 secondsfor the light chain and 150 seconds for the heavy chain.

[Reaction Conditions]

30 cycles at 94° C. for 2 minutes, 98° C. for 10 seconds, 50° C. for 30seconds and 68° C. for Y seconds, and at 68° C. for 3 minutes.

Using the 5′RACE product obtained in the above reaction, a solutionhaving the following composition was prepared. The solution was reactedat 60° C. for 30 minutes, and adenine was added to the end of the 5′RACEproduct.

5′RACE product 9 μL 10x A-attachment mix 1 μL Total 10 μL

A TA cloning reaction solution having the following composition wasprepared using the resulting adenine addition product and pcDNA (tradename) 3.4 TOPO (registered trademark) TA cloning kit. The reactionsolution was incubated at room temperature for 10 minutes, and theadenine adduct was cloned into pcDNA3.4.

[TA Cloning Reaction Solution]

Adenine adduct 4 μL salt solution 1 μL pCDNA3.4 1 μL Total 6 μL

(1.1.3) Transformation, Plasmid Extraction and Sequence Confirmation

The TA cloning sample (3 μL) obtained in the above (1.1.2) was added toDH5α (30 μL), and the mixture was allowed to stand on ice for 30minutes. Thereafter, the mixture was heat shocked by heating at 42° C.for 45 seconds. The mixture was again allowed to stand on ice for 2minutes, then the whole amount was applied to an ampicillin-containingLB plate. The plate was incubated at 37° C. for 16 hours. Singlecolonies on the plate were placed in the ampicillin-containing LB liquidmedium, and the medium was shake-cultured (250 rpm) at 37° C. for 16hours. The culture was centrifuged at 5000×G for 5 minutes to recover E.coli transformants. Plasmids were extracted from the recovered E. coliusing the QIAprep Spin Miniprep kit. Specific operations were carriedout according to the manual attached to the kit. The base sequence ofthe obtained plasmid was confirmed using pcDNA3.4 vector primer.Hereinafter, this plasmid was used as a plasmid for expressing mammaliancells.

(1.2) Acquisition of Wild-Type Anti-TSHR Antibody Gene

Synthesis of wild-type human anti-TSHR antibody gene was entrusted toGenScript Japan Inc. to obtain the wild-type human anti-TSHR antibodygene.

(2) Acquisition of Genes of Variants of Each Antibody (2.1) PrimerDesign and PCR

In order to introduce a mutation in FR3 defined by the Chothia method inthe light chain of each antibody, PCR was carried out using the plasmidcontaining the wild-type anti-insulin antibody gene obtained in theabove (1.1.3), the wild-type anti-TSHR antibody gene obtained in theabove (1.2), and the primer represented by the following base sequence.A D5 variant is a variant in which 5 amino acid residues of FR3 aremutated to aspartic acid residues, a E5 variant is a variant in which 5amino acid residues of FR3 are mutated to glutamic acid residues, a K5variant is a variant in which 5 amino acid residues of FR3 are mutatedto lysine residues, a R5 variant is a variant in which 5 amino acidresidues of FR3 are mutated to arginine residues, and a R3 variant is avariant in which 3 amino acid residues of FR3 are mutated to arginineresidues.

[Primer of anti-Insulin Antibody] Sequence 1 D5 Variant REV:(SEQ ID NO: 1) 5′ TTCGTATTCGGTCCCTTCCCCTTCGCCTTCAAAGCGAGCA 3′Sequence 2 E5 Variant REV: (SEQ ID NO: 2)5′ ATCGTAATCGGTCCCATCCCCATCGCCATCAAAGCGAGCA 3′Sequence 3 K5 Variant REV: (SEQ ID NO: 3)5′ CTTGTACTTGGTCCCCTTCCCCTTGCCCTTAAAGCGAGCA 3′Sequence 4 R5 Variant REV: (SEQ ID NO: 4)5′ TCTGTATCTGGTCCCTCTCCCTCTGCCTCTAAAGCGAGCA 3′ Sequence 5 FOR:(SEQ ID NO: 5) 5′ CTCACAATCAGCTGATTG 3′ Sequence 6 R3 Variant REV:(SEQ ID NO: 6) 5′ TCTCCCTCTGCCTCTAAAGCGAGCA 3′Sequence 7 R3 Variant FOR: (SEQ ID NO: 7) 5′ GGGACCAGATACAGA 3′

The primer of Sequence 5 was used as a forward primer common to theprimers of Sequences 1 to 4. The primer of Sequence 7 was used as aforward primer for the primer of Sequence 6.

[Primer of Anti-TSHR Antibody] Sequence 8 D5 Variant FOR: (SEQ ID NO: 8)5′ GGCACAGACGCCGACCTGGCAATCA 3′ Sequence 9 D5 Variant REV:(SEQ ID NO: 9) 5′ GTCCCGGTCTCCGTCAAACCGGTCG 3′Sequence 10 E5 Variant FOR: (SEQ ID NO: 10)5′ GGCACAGAGGCCGAGCTGGCAATCA 3′ Sequence 11 E5 Variant REV:(SEQ ID NO: 11) 5′ CTCCCGCTCTCCCTCAAACCGGTCG 3′Sequence 12 K5 Variant FOR: (SEQ ID NO: 12)5′ GGCACAAAGGCCAAGCTGGCAATCA 3′ Sequence 13 K5 Variant REV:(SEQ ID NO: 13) 5′ CTTCCGCTTTCCCTTAAACCGGTCG 3′Sequence 14 R5 Variant FOR: (SEQ ID NO: 14)5′ GGCACAAGGGCCAGGCTGGCAATCA 3′ Sequence 15 R5 Variant REV:(SEQ ID NO: 15) 5′ CCTCCGCCTTCCCCTAAACCGGTCG 3′

Using the plasmid obtained in the above (1.3) as a template, a PCRreaction solution having the following composition was prepared.

[PCR Reaction Solution]

10x PCR buffer 5 μL 25 mM Mg₂SO₄ 3 μL 2 mM dNTP mix 5 μL Forward primer1 μL Reverse primer 1 μL Template plasmid (40 ng/μL) 0.5 μL KOD plus neo(1 U/μL) 1 μL Purified water 33.5 μL Total 50 μL

The prepared PCR reaction solution was subjected to a PCR reaction underthe following reaction conditions.

[Reaction Conditions]

30 cycles at 98° C. for 2 minutes, 98° C. for 10 seconds, 54° C. for 30seconds and 68° C. for 4 minutes, and at 68° C. for 3 minutes.

The obtained PCR product was fragmented by adding 2 μL of DpnI (10 U/μL)to the PCR product (50 μL). Using the DpnI-treated PCR product, aligation reaction solution having the following composition wasprepared. The reaction solution was incubated at 16° C. for 1 hour toperform a ligation reaction.

[Ligation Reaction Liquid]

DpnI-treated PCR product 2 μL Ligation high ver. 2 5 μL T4Polynucleotide kinase 1 μL Purified water 7 μL Total 15 μL

(2.2) Transformation, Plasmid Extraction and Sequence Confirmation

A solution (3 μL) after the ligation reaction was added to DH5α (30 μL),and E. coli transformants were obtained in the same manner as in theabove (1.1.3). Plasmids were extracted from the obtained E. coli usingthe QIAprep Spin Miniprep kit. The base sequence of each obtainedplasmid was confirmed using pcDNA 3.4 vector primer. Hereinafter, theseplasmids were used as plasmids for expressing mammalian cells.

(3) Expression in Mammalian Cells [Reagents]

Expi293 (trademark) cells (Invitrogen)

Expi293 (trademark) Expression medium (Invitrogen)

ExpiFectamine (trademark) 293 transfection kit (Invitrogen)

(3.1) Transfection

Expi293 cells were proliferated by shaking culture (150 rpm) at 37° C.in a 5% CO₂ atmosphere. 30 mL of cell culture (3.0×10⁶ cells/mL) wasprepared according to the number of samples. A DNA solution of thefollowing composition was prepared using a plasmid encoding each variantof FR3 and a plasmid encoding a wild-type antibody. The DNA solution wasallowed to stand for 5 minutes.

[DNA Solution]

Light chain plasmid solution Amount (μL) corresponding to 15 μg Heavychain plasmid solution Amount (μL) corresponding to 15 μg Opti-MEM(trademark) Appropriate amount (mL) Total 1.5 mL

A transfection reagent having the following composition was prepared.The transfection reagent was allowed to stand for 5 minutes.

ExpiFectamine reagent 80 μL Plasmid solution 1420 μL Total 1.5 mL

The prepared DNA solution and the transfection reagent were mixed. Themixture was allowed to stand for 20 minutes. The resulting mixture (3mL) was added to the cell culture (30 mL). The mixture wasshake-cultured (150 rpm) at 37° C. for 20 hours in a 5% CO₂ atmosphere.After 20 hours, 150 μL and 1.5 mL of ExpiFectamine (trademark)transfection enhancers 1 and 2 were added to each culture, respectively.Each mixture was shake-cultured (150 rpm) at 37° C. for 6 days in a 5%CO₂ atmosphere.

(3.2) Recovery and Purification of Antibody

Each cell culture was centrifuged at 3000 rpm for 5 minutes, and theculture supernatant was recovered. The culture supernatant contains eachantibody secreted from transfected Expi293 (trademark) cells. Theobtained culture supernatant was again centrifuged at 15000×G for 10minutes, and the supernatant was recovered. To the resulting supernatant(30 mL) was added 100 μL of the antibody purification carrier Ab-CapcherMag (ProteNova), and the mixture was reacted at room temperature for 2hours. The carrier was magnetically collected to remove the supernatant,and PBS (1 mL) was added to wash the carrier. 400 μL of 100 mM Gly-HCl(pH 2.8) was added to the carrier, and the antibody (IgG) captured onthe carrier was eluted. This elution operation was performed three timesin total. The resulting eluate was neutralized with 100 mM Tris-HCl (pH8.0) to obtain an antibody solution.

(4) Results

An antibody in which the 63rd, 65th, 67th, 70th and 72nd serine residuesof the light chain FR3 defined by the Chothia method in the wild-typeanti-insulin antibody and the wild-type anti-TSHR antibody weresubstituted with charged amino acid residues (aspartic acid residues,glutamic acid residues, lysine residues or arginine residues) wasobtained. An antibody in which the 63rd, 65th and 67th serine residuesof the light chain FR3 defined by the Chothia method in the wild-typeanti-insulin antibody were substituted with charged amino acid residues(arginine residues) was obtained.

Example 2 Measurement of Affinity of Antibody into which Charged AminoAcid Residue Introduced in FR3

How the affinity of each variant prepared in Example 1 for an antigenchanges as compared to that of the wild type was examined.

(1) Antibody Fragmentation

Using Pierce (trademark) Mouse IgGl Fab and F(ab′)2 Preparation kit(Thermo Fisher), each antibody obtained in Example 1 was made into Fabfragments. Specific operations were carried out according to the manualattached to the kit. The resulting reaction solution was subjected togel filtration purification using Superdex 200 Increase 10/300 GL (GEHealthcare). The 50 kDa elution fraction was collected, and the obtainedfraction was used as a Fab fragment-containing solution for subsequentexperiments.

(2) Measurement of Affinity (2.1) Measurement of Affinity by SPRTechnique

The affinity of wild-type anti-insulin antibody and its variant for anantigen was measured by SPR technique as follows. Humulin R U-100 (EliLilly) was used as an antigen for anti-insulin antibody. Antigen wasimmobilized (immobilization: 100 RU) to a sensor chip for Biacore(registered trademark) Series S Sensor Chip CM5 (GE Healthcare). 50 nM,25 nM, 12.5 nM, 6.25 nM and 3.13 nM solutions were prepared by dilutingthe Fab fragment-containing solution of the anti-insulin antibody. Fabfragment-containing solutions at each concentration were delivered toBiacore (registered trademark) T200 (GE Healthcare) (association time of120 seconds and dissociation time of 1200 seconds). Measurement data wasanalyzed using Biacore (registered trademark) Evaluation software, andthe data on the affinity of anti-insulin antibody was obtained.

(2.2) Evaluation of Affinity by ELISA Method

The affinity of wild-type anti-TSHR antibody and its variant for anantigen was measured by ELISA method as follows.

(2.2.1) Immobilization of Capture Antibody

As a capture antibody, 4E31 antibody (RSR Limited), which was a mousemonoclonal anti-TSHR antibody, was used. The 4E31 antibody (5 μg) wasdiluted with PBS to obtain an antibody solution. 100 μL each of thisantibody solution was added to each well of NUNC-immuno module (Cat No.469949, manufactured by NUNC, hereinafter referred to as “plate”). Thisplate was allowed to stand at room temperature for 3 hours to immobilizethe 4E31 antibody on the well. The antibody solution was removed, and300 μL each of a blocking solution (PBS containing 1% BSA) was added toeach well of the plate. Blocking was performed at 4° C. for 20 hours ormore.

(2.2.2) Primary Reaction

Detergent solubilized cell membrane preparation containing the TSHR (RSRLimited) was used as the antigen of the anti-TSHR antibody. This antigenwas diluted 500-fold with PBS containing 1% BSA to obtain an antigensolution. The blocking solution was removed from the plate on which the4E31 antibody was immobilized, and 50 μL each of the antigen solutionwas added to each well. This plate was shaken at room temperature for 60minutes to perform an antigen-antibody reaction.

(2.2.3) Secondary Reaction

As detection antibodies, a wild-type anti-TSHR antibody, a D5 variantand a R5 variant were used. Each antibody was stepwise diluted with PBScontaining 1% BSA to obtain antibody solutions at concentrations of 1000pM, 100 pM, 10 pM, 1 pM, and 0.1 pM. HRP-labeled anti-human IgG (Fcspecific) antibody was used as a secondary antibody. This secondaryantibody was diluted with PBS containing 1% BSA to obtain a secondaryantibody solution at a concentration of 0.2 μg/mL. The antibody solution(50 μL) of each concentration and the secondary antibody solution (50μL) were mixed to obtain a mixed solution of antibodies. The antigensolution was removed from the plate, and 300 μL each of a washingsolution (PBS containing 1% BSA) was added to each well. Then, thewashing solution was removed from the plate, and 300 μL each of washingsolution was added to each well for washing. This washing operation wasrepeated three times. The washing solution was removed from the plate,and 100 μL each of the mixed solution of antibodies was added to eachwell. This plate was shaken at room temperature for 60 minutes toperform an antigen-antibody reaction. After the reaction, the abovewashing operation was repeated three times.

(2.2.4) Detection

As a substrate solution, 1-Step Ultra TMB-ELISA Substrate Solution(Thermo Fisher Scientific) was used. The washing solution was removedfrom the plate, and the substrate solution was added at 100 μL/well.This plate was allowed to stand at room temperature for 5 minutes. After5 minutes, 100 μL each of a stop solution (0.1 M H₂SO₄) was added toeach well of the plate to terminate the reaction. Then, the absorbanceat 450 nm was measured for each well of the plate.

(3) Results

The dissociation constant (K_(D)) was calculated from the binding rateconstant (k_(on)) and the dissociation rate constant (k_(off)) obtainedfor the anti-insulin antibody. The dissociation constant (K_(D)) wascalculated from the measurement value of the ELISA method using theanti-TSHR antibody. The kinetic parameters of each antibody are shown inTables 3 and 4, and FIGS. 1A and 1B. In the figures, “positivelycharged” indicates the K_(D) value of the R5 variant, and “negativelycharged” indicates the K_(D) value of the D5 variant. In Table 3, thevalues obtained by global fitting are “average value±standard error”.

TABLE 3 Anti-insulin antibody Sample K_(D) (M) K_(on) (M⁻¹s⁻¹) K_(off)(s⁻¹) Wild type 2.22 × 10⁻¹⁰ (2.48 ± 0.01) × 10⁶ (5.49 ± 0.01) × 10⁻⁴ R3Variant 5.49 × 10⁻¹¹ (8.79 ± 0.02) × 10⁶ (4.83 ± 0.01) × 10⁻⁴ R5 Variant2.03 × 10⁻¹² (7.19 ± 0.05) × 10⁶ (1.46 ± 0.01) × 10⁻⁵ K5 Variant 8.04 ×10⁻¹² (5.44 ± 0.04) × 10⁷ (4.37 ± 0.01) × 10⁻⁴ D5 Variant 9.69 × 10⁻¹⁰(7.64 ± 0.04) × 10⁵ (7.40 ± 0.02) × 10⁻⁴ E5 Variant 1.52 × 10⁻⁹  (2.79 ±0.01) × 10⁵ (4.24 ± 0.01) × 10⁻⁴

TABLE 4 Anti-TSHR antibody Sample K_(D) (M) Wild type 7.0 × 10⁻¹⁰ D5Variant 3.0 × 10⁻⁸  R5 Variant 6.5 × 10⁻¹⁰

From Table 3 and FIG. 1A, the K_(D) values of the R3 variant, the R5variant and the K5 variant of the anti-insulin antibody were lower thanthe K_(D) value of the wild type. The K_(D) values of the D5 variant andthe E5 variant were higher than the K_(D) value of the wild type.Therefore, as to the anti-insulin antibody, it was found that anantibody with affinity for an antigen improved as compared to the wildtype can be prepared by mutating 3 or 5 amino acid residues of FR3 tobasic amino acid residues. It was found that an antibody with affinityfor an antigen reduced as compared to the wild type can be prepared bymutating 5 amino acid residues of FR3 to acidic amino acid residues.

From Table 4 and FIG. 1B, the K_(D) value of the D5 variant of theanti-TSHR antibody was higher than the K_(D) value of the wild type. Asto the anti-TSHR antibody, it was found that an antibody with affinityfor an antigen reduced as compared to the wild type can be prepared bymutating 5 amino acid residues of FR3 to acidic amino acid residues. Onthe other hand, the K_(D) value of the R5 variant of the anti-TSHRantibody was comparable to the K_(D) value of the wild type. That is, asto the anti-TSHR antibody, it is suggested that affinity does not changeeven when 5 amino acid residues of FR3 are mutated to basic amino acidresidues.

Example 3 Relationship Between Electrical Characteristic of Amino AcidSequence of CDR and Affinity for Antigen

From Example 2, as to the anti-insulin antibody, affinity could beimproved and reduced by introducing mutation in FR3. On the other hand,as to the anti-TSHR antibody, affinity could be reduced even byintroducing mutation in FR3, but affinity could not be improved.Therefore, the influence of the antigen-binding site of the antibody onthe surface charge by introducing mutation in FR3 was examined.

(1) Study of Change in Antibody Surface Charge by Mutation Introduction

The surface charge distribution of various Fab fragments prepared inExample 1 was analyzed using a Discovery Studiou (Dassault SystèmesBIOVIA). The surface charge distribution diagrams of the Fab fragment ofthe anti-insulin antibody and insulin as an antigen are shown in FIG.2A. The surface charge distribution diagrams of the Fab fragment of theanti-TSHR antibody and TSHR as an antigen are shown in FIG. 2B.

In the figure, the arrow indicates the antigen-binding site, and PIindicates the value of the isoelectric point. Here, the antigen-bindingsite is the same as CDR. In the figure, the surface charge distributionis shown in color, indicating that the blue portion is positivelycharged, the red portion is negatively charged, and the white portion iselectrically neutral.

From FIG. 2A, it was found that, in the wild-type anti-insulin antibody,the surface charge of the antigen-binding site is neutral. In thevariant type in which a basic amino acid residue was introduced(positively charged mutation) in FR3, a wide range of surface chargesincluding the antigen-binding site was positive. Here, insulin as anantigen is a negatively charged protein, and from FIG. 1A, the affinityfor a variant-type antigen in which a positively charged mutation wasintroduced was improved. On the other hand, in the variant type in whichan acidic amino acid was introduced (negatively charged variation) inFR3, a wide range of surface charges including the antigen-binding sitewas negative. From FIG. 1A, the affinity for the variant-type antigen inwhich the negatively charged mutation was introduced was reduced. Fromthese facts, it is understood that, in the wild-type anti-insulinantibody, the contribution to the surface charge by charged amino acidresidues introduced in FR3 spreads over a wide range.

From FIG. 2B, it was found that, in the wild-type anti-TSHR antibody,the surface charge of the antigen-binding site is negative. In thevariant type in which a basic amino acid was introduced (positivelycharged variation) in FR3, the surface charge in the range excluding theantigen-binding site was positive, but the surface charge of theantigen-binding site did not change much. Here, TSHR as an antigen is anegatively charged protein, but from FIG. 1B, there was no change inaffinity for a variant-type antigen in which a positively chargedmutation was introduced. On the other hand, in the variant type in whichan acidic amino acid was introduced (negatively charged variation) inFR3, a wide range of surface charges including the antigen-binding sitewas negative. From FIG. 1B, the affinity for the variant-type antigen inwhich the negatively charged mutation was introduced was reduced. Fromthese facts, it is understood that, in the wild-type anti-TSHR antibody,the contribution to the surface charge by acidic amino acid residuesintroduced in FR3 spreads over a wide range. On the other hand, evenwhen a basic amino acid residue is introduced in FR3 of the wild-typeanti-TSHR antibody, it is understood that the surface charge is locallydifferent.

(2) Relationship between Electrical Characteristic of Amino AcidSequence of CDR and Control of Affinity

The present inventors considered that the electrical characteristic ofCDR of the antibody is related to how the affinity for an antigenchanges by introduction of charged amino acid residues in FR3 of theantibody. Here, the present inventors defined the electricalcharacteristic of CDR by the following formula (I).

X=[Number of basic amino acid residues in amino acid sequence ofCDR]−[Number of acidic amino acid residues in amino acid sequence ofCDR]  (I)

wherein when X is −1, 0 or 1, the electrical characteristic of CDR isneutral,

when X is 2 or more, the electrical characteristic of CDR is positivelycharged, and

when X is −2 or less, the electrical characteristic of CDR is negativelycharged.

Table 5 shows the amino acid sequence of the light chain CDR of thewild-type anti-TSHR antibody (SEQ ID NOs: 16 and 17). The amino acidsequences of these CDRs are sequences defined by the Chothia method.

TABLE 5 Electrical characteristic Antibody CDR1 CDR2 CDR3 of CDRAnti-TSHR antibody GNSSNIGNNA YDD WDDSLDSQ Negatively charged

The CDR of the anti-insulin antibody has one basic amino acid residue(arginine), and no acidic amino acid residue exists, thus the electricalcharacteristic of CDR is defined as neutral (X=1). As shown in Table 5,the CDR of the anti-TSHR antibody has five acidic amino acid residues(aspartic acid), and no basic amino acid residue exists, thus theelectrical characteristic of CDR is defined as negatively charged(X=−5). As can be seen from FIGS. 2A and 2B, the electricalcharacteristics of the CDRs of the anti-insulin antibody and theanti-TSHR antibody determined by the formula (I) are consistent with thesurface charge of the antigen-binding site analyzed by DiscoveryStudiou. Thus, it was found that there are biases in the electricalcharacteristic of CDR and the surface charge of the antigen-bindingsite, depending on the antibody.

(3) Results

From the analysis of Example 2 and Example 3, it is suggested that, inthe antibody whose electrical characteristic of CDR is neutral, acontribution of the introduction of charged amino acid residue in FR3 islarge. It is suggested that, in the antibody whose electricalcharacteristic of CDR is neutral, it is possible to control theorientation of the antigen-binding site by electrostatic interactioncaused by the introduction. On the other hand, it is suggested that, inthe antibody whose electrical characteristic of CDR is negativelycharged, the effect of electrostatic interaction is topical, even when abasic amino acid residue is introduced in FR3. However, it is suggestedthat, in the antibody whose electrical characteristic of CDR isnegatively charged, it is possible to reduce affinity for an antigen byelectrostatic repulsive force when introducing an acidic amino acidresidue in FR3.

Example 4 Study on Thermal Stability of Antibody into which ChargedAmino Acid Residue Introduced in FR3

How the thermal stability of each variant of the anti-insulin antibodyprepared in Example 1 changes as compared to that of the wild type wasexamined.

(1) Substitution of Buffer by Gel Filtration

The solvent of the Fab fragment-containing solution obtained in Example2 was substituted with a buffer (phosphate buffered saline: PBS) usedfor measurement with a differential scanning calorimeter (DSC) by gelfiltration. The conditions of gel filtration are as follows.

[Conditions of Gel Filtration]

Buffer: PBS

Column used: Superdex 200 Increase 10/300 (GE Healthcare)

Column volume (CV): 24 mL

Sample volume: 500 μL

Flow rate: 1.0 mL/min

Elution amount: 1.5 CV

Fraction volume: 500 μL

(2) Measurement of Denaturation Temperature (Tm)

Fractions containing Fab fragments were diluted with PBS to prepare Fabfragment-containing samples (final concentration 5 μM). Tm of each Fabfragment was measured using MicroCal VP-Capillary DSC (MalvernInstruments Ltd). The measurement conditions are as follows.

[DSC Measurement Conditions]

Sample amount: 400 μL

Measurement range: 30° C. to 90° C.

Heating rate: 60° C./hour

(3) Results

The Tm value and analytical peak obtained by DSC measurement are shownin Table 6 and FIG. 3, respectively.

TABLE 6 Sample Tm (° C.) Wild type 76.8 ± 0.087 D5 Variant 66.9 ± 0.046E5 Variant 69.5 ± 0.071 R5 Variant 71.1 ± 0.150 R3 Variant 74.4 ± 0.069

The D5 variant showed the lowest thermal stability as compared to thewild type, but the reduction remained only around 13%. In most variants,the thermal stability was found to be almost unchanged from that of thewild type. Thus, it is suggested that the introduction of charged aminoacid residue in FR3 hardly affects on the thermal stability of theantibody.

Example 5 Control of Affinity of anti-Lysozyme Antibody for Antigen

A mutation was introduced in FR3 of an anti-lysozyme antibody based onthe electrical characteristic of CDR, and the affinity of the obtainedvariant for lysozyme was confirmed.

(1) Electrical Characteristic of CDR of Anti-lysozyme Antibody

Synthesis of anti-lysozyme antibody gene was entrusted to GenScriptJapan Inc. to obtain a plasmid DNA containing wild-type anti-lysozymeantibody gene. Based on the base sequence of the gene, the amino acidsequence of the anti-lysozyme antibody was determined. Table 7 shows theamino acid sequences of the light chain CDR and heavy chain CDR of thewild-type anti-lysozyme antibody (SEQ ID NOs: 18 to 23). The amino acidsequences of these CDRs are sequences defined by the Chothia method.

TABLE 7 CDR1 CDR2 CDR3 Light RASQSIGNNLH YASQSIS QQSNSWPYT chain HeavySDYWS YVSYSGSTYYNPSLKS WDGDY chain

As shown in Table 7, the light chain CDR and heavy chain CDR of theanti-lysozyme antibody have two basic amino acid residues and threeacidic amino acid residues. Therefore, the electrical characteristic ofCDR of the anti-lysozyme antibody are defined as neutral (X=−1).

(2) Preparation of Variant of Anti-lysozyme Antibody

Synthesis of genes of R5 variant and D5 variant of anti-lysozymeantibody was entrusted to GenScript Japan Inc. to obtain a plasmid DNAcontaining variant genes of anti-lysozyme antibody. Here, the R5 variantof the anti-lysozyme antibody is an antibody in which the 63rd, 65th and67th serine residues, the 70th aspartic acid residue and the 72ndthreonine residue of the light chain FR3 defined by the Chothia methodin the wild-type antibody are substituted with arginine residues. The D5variant of the anti-lysozyme antibody is an antibody in which the 63rd,65th and 67th serine residues, the 70th aspartic acid residue and the72nd threonine residue of the light chain FR3 defined by the Chothiamethod in the wild-type antibody are substituted with aspartic acidresidues.

Using the obtained plasmid, each antibody was expressed in Expi293(trademark) cells, and the resulting culture supernatant was purified inthe same manner as in Example 1 to obtain each solution of wild-type, R5variant and D5 variant of anti-lysozyme antibodies.

(3) Measurement of Affinity of Variant for Antigen

A solution (200 ng/mL) prepared by dissolving hen egg white-derivedlysozyme (Sigma-Aldrich) in a 10 mM sodium acetate solution (pH 5.0) wasused as an antigen of the anti-lysozyme antibody. The antigen wasimmobilized on the sensor chip for Biacore (registered trademark) SeriesS Sensor Chip CMS (GE Healthcare) (41 RU or 33 RU). Solutions of variousconcentrations were prepared by diluting each antibody solution with HBSEP+buffer (GE Healthcare). These solutions were sent to Biacore(registered trademark) T200 (GE Healthcare). The antibody concentrationsand measurement conditions in each solution are as follows. Measurementdata was analyzed using Biacore (registered trademark) Evaluationsoftware, and the data on the affinity of each antibody was obtained.

[Antibody Concentrations]

Wild type: 30 nM, 15 nM, 7.5 nM, 3.75 nM and 1.875 nM

D5 variant: 30 nM, 15 nM, 7.5 nM, 3.75 nM and 1.875 nM

R5 variant: 2 nM, 1 nM, 0.5 nM, 0.25 nM and 0.125 nM

[Measurement Conditions]

Association: 30 μL/min, 60 sec, 120 sec

Dissociation: 30 μL/min, 60 sec, 1200 sec

Regeneration: Gly-HCl (pH 2.0)/60 μL/min, 40 sec

(4) Results

The dissociation constant (K_(D)) was calculated from the binding rateconstant (k_(on)) and the dissociation rate constant (k_(off)) obtainedfor the wild-type and variants of anti-lysozyme antibodies. The kineticparameters of each antibody are shown in Table 8 and FIG. 4. In thefigure, “negatively charged” indicates the K_(D) value of the D5variant, and “positively charged” indicates the K_(D) value of the R5variant.

TABLE 8 Sample K_(D) (M) K_(on) (M⁻¹s⁻¹) K_(off) (s⁻¹) Wild type 2.34 ×10⁻¹⁰ 4.88 × 10⁵ 1.14 × 10⁻⁴ D5 Variant 4.50 × 10⁻¹⁰ 6.07 × 10⁵ 2.73 ×10⁻⁴ (negatively charged) R5 Variant 2.81 × 10⁻¹² 3.34 × 10⁷ 9.40 × 10⁻⁵(positively charged)

From Table 8 and FIG. 4, the K_(D) value of the R5 variant of theanti-lysozyme antibody was lower than the K_(D) value of the wild type.The K_(D) value of the D5 variant was higher than the K_(D) value of thewild type. Therefore, as to the anti-lysozyme antibody, it was foundthat an antibody with affinity for an antigen improved as compared tothe wild type can be prepared by mutating 5 neutral amino acid residuesof FR3 to basic amino acid residues. It was found that an antibody withaffinity for an antigen reduced as compared to the wild type can beprepared by mutating 5 neutral amino acid residues of FR3 to acidicamino acid residues. These results were similar to those of the variantof the anti-insulin antibody in Example 2. Therefore, it is suggestedthat, in the antibody whose electrical characteristic of CDR is neutral,it is possible to control the orientation of the antigen-binding site byelectrostatic interaction caused by the introduction of a charged aminoacid residue in FR3.

Example 6 Control of Affinity of Anti-HBsAg Antibody for Antigen

A mutation was introduced in FR3 of an anti-HBsAg antibody based on theelectrical characteristic of CDR, and the affinity of the obtainedvariant for lysozyme was confirmed.

(1) Electrical Characteristic of CDR of Anti-HBsAg Antibody

Hybridomas that produce a mouse anti-HBsAg antibody were prepared byusing recombinant HBsAg as an antigen, according to the method describedin Kohler and Milstein, Nature, vol. 256, p. 495-497, 1975. A plasmidDNA containing the wild-type anti-HBsAg antibody gene was obtained fromRNA of the hybridoma in the same manner as in Example 1. Based on thebase sequence of the gene, the amino acid sequence of the anti-HBsAgantibody was determined. It was found that the light chain CDR and heavychain CDR defined by the Chothia method in the wild-type anti-HBsAgantibody had two basic amino acid residues and ten acidic amino acidresidues. Therefore, the electrical characteristic of CDR of theanti-HBsAg antibody are defined as negatively charged (X=−8).

(2) Preparation of Variant of Anti-HBsAg Antibody

In order to introduce a mutation in the light chain FR3 defined by theChothia method, PCR was carried out in the same manner as in Example 1,using the wild-type anti-HBsAg antibody gene obtained in the above (1)and the primer represented by the following base sequence.

[Primer of anti-HBsAg Antibody] D5 variant FOR: (SEQ ID NO: 24)5′ GGGACCGATTATGATCTCACAATCAGTCGAATGGAG 3′ D5 variant REV:(SEQ ID NO: 25) 5′ ATCCCCATCGGCATCGAAACGAACAGGGACTCCAGAAGC 3′

Using the obtained PCR product, a plasmid containing a gene encoding avariant or wild-type light chain and a plasmid containing a geneencoding a wild-type heavy chain were obtained in the same manner as inExample 1. Using these plasmids, each antibody was expressed in Expi293(trademark) cells, and the resulting culture supernatant was purified inthe same manner as in Example 1 to obtain each solution of wild-type andD5 variant of anti-HBsAg antibodies. Here, the D5 variant of theanti-HBsAg antibody is an antibody in which the 63rd, 65th, 67th and70th serine residues and the 72nd phenylalanine residue of the lightchain FR3 defined by the Chothia method in the wild-type antibody aresubstituted with aspartic acid residues.

(3) Measurement of Affinity of Variant for Antigen (3.2) Immobilizationof Capture Antibody

The capture antibody was immobilized on each well of a plate(NUNC-immuno module, Cat No. 469949, manufactured by NUNC Co., Ltd.) inthe same manner as in Example 2, except for using a mouse anti-HBsAgantibody produced from a hybridoma different from the hybridoma obtainedin the above (1) as a capture antibody. Each well of the plate wasblocked with a blocking solution (PBS containing 1% BSA) in the samemanner as in Example 2.

(3.3) Primary Reaction

As the antigen of the anti-HBsAg antibody, HISCL (registered trademark)HBsAg calibrator (HBsAg concentration 0.025 IU/mL, Sysmex Corporation)was used. The blocking solution was removed from the plate on which thecapture antibody was immobilized, and 50 μL each of the antigen solutionwas added to each well. This plate was shaken at room temperature for 60minutes to perform an antigen-antibody reaction.

(3.3) Secondary Reaction and Detection

The wild-type and D5 variant of anti-HBsAg antibodies were used asdetection antibodies. Each antibody was stepwise diluted with PBScontaining 1% BSA to obtain antibody solutions at concentrations of 400nM, 80 nM, 16 nM, 3.2 nM, 640 pM, 128 pM, 25.6 pM and 5.12 pM.HRP-labeled anti-mouse IgG (Fc specific) antibody was used as asecondary antibody. Using these, an antigen-antibody reaction wasperformed in the same manner as in Example 2. Then, the absorbance at450 nm was measured for each well of the plate using 1-Step UltraTMB-ELISA Substrate Solution (Thermo Fisher Scientific) as a substratesolution in the same manner as in Example 2.

(4) Results

The dissociation constant (K_(D)) was calculated from the measurementvalue of the ELISA method using the wild-type and D5 variant ofanti-HBsAg antibodies. The results are shown in Table 9 and FIG. 5. Inthe figure, “negatively charged” indicates the K_(D) value of the D5variant.

TABLE 9 Sample K_(D) (M) Wild type 2.00 × 10⁻¹⁰ D5 Variant (negativelycharged) 3.00 × 10⁻⁹ 

From Table 9 and FIG. 5, the K_(D) value of the D5 variant of theanti-HBsAg antibody was higher than the K_(D) value of the wild type.The K_(D) value of the D5 variant was higher than the K_(D) value of thewild type. Therefore, as to the anti-HBsAg antibody, it was found thatan antibody with affinity for an antigen reduced as compared to the wildtype can be prepared by mutating 5 neutral amino acid residues of FR3 toacidic amino acid residues. These results were similar to those of theD5 variant and the E5 variant of the anti-TSHR antibodies in Example 2.Therefore, it is suggested that, in the antibody whose electricalcharacteristic of CDR is negatively charged, it is possible to reduceaffinity for an antigen by electrostatic repulsive force generated bythe introduction of an acidic amino acid residue in FR3.

1-20. (canceled)
 21. An antibody comprising a light chain and a heavychain, comprising complementarity-determining regions (CDRs) which havean electrical characteristic value (X) of −1, 0 or 1, wherein at least 3amino acid residues selected from the group consisting of positions 63,65, 67, 70 and 72 in the light chain as defined by the Chothia methodeach independently is an arginine residue or a lysine residue, and X iscalculated by the following formula:X=[total number of basic amino acid residues in all CDRs in theantibody]−[total number of acidic amino acid residues in all CDRs in theantibody], wherein the CDRs are defined by the Chothia method.
 22. Theantibody according to claim 21, wherein the antibody is in the form ofan antibody fragment or an IgG.
 23. The antibody according to claim 22,wherein the antibody fragment is a Fab fragment.
 24. The antibodyaccording to claim 21, wherein said at least 3 amino acid residuescomprises amino acid residues at the positions 63, 65, and 67 in thelight chain as defined by the Chothia method.
 25. The antibody accordingto claim 21, wherein amino acid residues at the positions 63, 65, 67, 70and 72 in the light chain as defined by the Chothia method eachindependently is an arginine residue or a lysine residue.
 26. Theantibody according to claim 21, wherein said at least 3 amino acidresidues are arginine residues.
 27. The antibody according to claim 21,wherein said at least 3 amino acid residues are lysine residues.
 28. Theantibody according to claim 21, wherein the antibody comprises CDR1,CDR2 and CDR3 in the light chain and CDR1, CDR2 and CDR3 in the heavychain, and X is calculated by the following formula:X=[total number of basic amino acid residues in 6 CDRs in theantibody]−[total number of acidic amino acid residues in 6 CDRs in theantibody].